Cholera toxin, the ADP-ribosyltransferase secreted by Vibrio cholerae, is a 84-kDa protein comprising one catalytic A subunit associated non-covalently with five B subunits.Through its B subunit pentamer, the toxin binds the cell surface receptor monosialoganglioside GM1, which facilitates entry of the A subunit and through the plasma membrane access to its intracellular substrates, beta-NAD and heterotrimeric G protein subunit alpha/s. Like other G proteins, Galpha/s is active with bound GTP. Its intrinsic GTPase activity converts the active GTP-bound protein to the inactive GDP-bound species. The Galpha/s catalytic arginine is critical for GTP hydrolysis to GDP and thereby inactivation. ADP-ribosylation of that arginine inhibits GTPase activity and prolongs the life-time of active, GTP-bound Galpha/s. Resulting persistent activation of adenylyl cyclase increases cyclic AMP accumulation, along with its effects on water and electrolyte transport that cause the diarrhea of cholera.[unreadable] We asked whether the ADP-ribosylarginine hydrolase, ARH1, can counteract the effects of cholera toxin on cells, by catalyzing the removal of ADP-ribose from the modified Galpha/s protein. To evaluate this possibility, we studied the small intestine (and other cells) from ARH1 knock-out mice, generated in our laboratory, that lack hydrolase activity. Sensitivity to cholera toxin was assessed by determining the extent of modification of Galpha/s, and the levels of ADP-ribosyl(arginine)protein. Cholera toxin effects on fluid accumulation in intestinal loops were much greater in knockout than wild-type mice. Data from experiments with cells grown from mouse embryos were similarly consistent with the conclusion that the hydrolase can, indeed, moderate the effects of intoxication both in vitro and in vivo. Thus, cholera toxin-catalyzed ADP-ribosylation of cell proteins can be counteracted by ARH1, which could function as a modifier gene in disease. Further, our studies demonstrate that enzymatic cross-talk exists between bacterial toxin ADP-ribosyltransferases and host ADP-ribosylation cycles. In disease, toxin-catalyzed ADP-ribosylation can overwhelm this potential host defense system, resulting in persistence of ADP-ribosylation and intoxication of the cell.[unreadable] [unreadable] In addition to the pathway described above, other ADP-ribosylation pathways exist in cells and are involved in the regulation of critical biological systems. Poly(ADP-ribosylation) involves the formation of long, branched chains of poly(ADP-ribose), in reactions catalyzed by a family of poly(ADP-ribose) polymerases. These enzymes participate in DNA repair, cell differentiation, and carcinogenesis. Poly-ADP-ribosylation is reversed by a poly(ADP-ribose) glycohydrolase (PARG). We had earlier identified an ARH1related protein, ARH3, which exhibited PARG-like activity, although the kcat was significantly lower than PARG. The silent information regulator 2 (Sir2) family of NAD-dependent N-acetyl-protein deacetylases participates in the regulation of gene silencing, chromatin structure, and longevity. In the Sir2-catalyzed reaction, the acetyl moiety of N-acetyl-histone is transferred to the ADP-ribose of NAD, yielding O-acetyl-ADP-ribose and nicotinamide. Some of these enzymes may, in addition, transfer ADP-ribose to proteins, similar to the activities of the ADP-ribosyltransferases described above. We hypothesized that if O-acetyl-ADP-ribose were an important signaling molecule, a specific hydrolase would cleave the (O-acetyl)-(ADP-ribose) linkage. We observed that the poly(ADP-ribose) glycohydrolase ARH3, hydrolyzed O-acetyl-ADP-ribose to produce ADP-ribose in a time- and Mg2+-dependent reaction and thus could participate in this signaling pathway as well as poly-ADP-ribosylation. Thus, O-acetyl-ADP-ribose hydrolase belongs to the ARH family of three, structurally related 39-kDa ADP-ribose-binding proteins (ARH1-3). ARH1 hydrolyzed ADP-ribosylarginine, whereas ARH3 degraded poly(ADP-ribose). ARH3 catalyzed the generation of ADP-ribose from O-acetyl-ADP-ribose significantly faster than from poly(ADP-ribose). Like the cleavage of poly(ADP-ribose) hydrolysis of O-acetyl-ADP-ribose by ARH3, was abolished by replacement of the vicinal aspartates at positions 77 and 78 of ARH3 with alanine. Thus, identical active site residues are crucial for both activities. The rate of O-acetyl-ADP-ribose hydrolysis by recombinant ARH3 was 250-fold that observed with ARH1; ARH2 and poly-ADP-ribose glycohydrolase were inactive. All data support the conclusion that the Sir2 reaction product, O-acetyl-ADP-ribose, is degraded by ARH3. Thus, we have demonstrated that ARH3 may be a critical participant in two important signal transduction pathways, poly(ADP-ribosylation) and protein de-acetylation.